WO1999025719A1

WO1999025719A1 - Novel physiologically active substance sulphostin, process for producing the same, and use thereof

Info

Publication number: WO1999025719A1
Application number: PCT/JP1998/005164
Authority: WO
Inventors: Tomio Takeuchi; Masa Hamada; Yasuhiko Muraoka; Tetsuo Akiyama; Masatoshi Abe; Hiroshi Naganawa; Yoshikazu Takahashi
Original assignee: Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai
Priority date: 1997-11-18
Filing date: 1998-11-17
Publication date: 1999-05-27
Also published as: US6214340B1; CA2310400A1; EP1043328A4; DE69839279D1; JP4095772B2; EP1043328B1; CA2310400C; DE69839279T2; EP1043328A1

Abstract

A physiologically active substance, sulphostin, having a potent dipeptidylpeptidase IV inhibitory activity and obtained by culturing a microorganism belonging to the genus Streptomyces and capable of producing the above substance to accumulate the same in the culture medium and harvesting the same from the medium.

Description

TECHNICAL FIELD The present invention relates to a novel physiologically active substance sulfostine, a method for producing the same, and a use thereof. The compounds of the present invention have a dipeptidyl peptidase IV inhibitory action. It is known that dipeptidyl peptidase IV present on the surface of T cells is involved in the activation of T cells (Immuno 1. Today, 15, 180-184 (1994)). Plays an important role in the immune system. Moreover, the di-peptidyl peptidase IV is involved in the degradation of the growth hormone releasing hormone in serum (J. C li n I nv es t, 83, 1 533 -.. 1 540 (1 989)) 0 was Thus, the compounds of the present invention are expected to be used as bioactive substances in pharmaceuticals such as immunomodulators, hormone modulators, anti-HIV drugs, anti-allergic drugs, anti-inflammatory drugs, and anti-rheumatic drugs. .

Background art

Heretofore, diprotin A and B (J. of Antibiotics 37, 422-425 (19894)) and the like have been known as physiologically active substances having dipeptidyl peptidase IVP and harmful effects.

However, these substances have a weak inhibitory effect on the enzyme, and were not satisfactory as physiologically active substances for the above-mentioned pharmaceutical uses. Therefore, the emergence of new compounds suitable for these uses has been awaited.

Disclosure of the invention

Accordingly, the present inventors have searched extensively for metabolites of microorganisms and found that a strain belonging to actinomycetes produces a physiologically active substance sulfostine having an excellent inhibitory activity against dipeptidyl peptidase IV. The present invention has been completed.

The physiologically active substance sulfostin of the present invention is used in Streptomyces

It can be obtained by culturing a sulfostine-producing bacterium belonging to the genus (S treptomyces), producing and accumulating the physiologically active substance in the culture, and collecting this from the culture. Brief description of drawings ·

Figure 1 shows the infrared absorption spectrum of sulfostine measured with a potassium bromide tablet.

Figure 2 shows the nuclear magnetic resonance spectrum of sulfostin measured in heavy water. Figure 3 is a carbon nuclear magnetic resonance spectrum of sulfostine measured in heavy water. Figure 4 shows the nuclear magnetic resonance spectrum of sulfostin measured in heavy water. BEST MODE FOR CARRYING OUT THE INVENTION

A representative example of sulfostine-producing bacteria was Streptomyces espi isolated from soil in Hamo-cho, Sado-gun, Niigata Prefecture in September 1994. — 43 F 3 shares (FE RM BP—657 No. 1, Institute of Biotechnology, Industrial Science and Technology, 305-8566, 1-3-1, Higashi 1-chome, Tsukuba, Ibaraki, Japan; commissioned on February 6, 1997 ). This strain is an example of a strain most effectively used in the present invention. The bacteriological and physiological properties of this strain are as follows.

1. Morphological properties

The MK 25 1-43 F 3 strain elongates aerial hyphae capable of forming a helix from the branched basic hyphae. The mature spore chain has from 10 to 50 or more oval spores, and the size of one spore is about 0.6-0.8 micron x about 0.8- 0.9 microns. The surface of the spores is prickly to hairy. No vegetative branches and fungal filaments are found in the cells, and neither spores nor motile spores are found.

2. Growth in various media

For the color of the colonies on the culture medium, after the general Japanese color name, [] is used as the color standard in the container, the color of the container, the color of the ブ b'America, the color of the color, and the color of the color. Use the colorha rmony ma nu al of Containaer Corporation of America to describe the color expression.

(1) Sucrose / nitrate agar (cultured at 27 ° C)

On colorless growth, white-brown [3 ba, Earl] aerial mycelium grows on the skin. No soluble dye is found. (2) Glycerin. Asparagine agar medium (ISP-culture medium 5, cultured at 27 ° C) Light yellow [2 cb, Ivory Tint], aerial mycelium does not form, and no soluble pigment is observed .

(3) Starch Inorganic salt agar medium (ISP—medium 4) (cultured at 27 ° C) Slightly white-brown aerial mycelium on colorless to pale yellow tea [2 gc, Bambo] To settle. No soluble dye is found.

(4) Tyrosine agar medium (ISP—medium 7) (cultured at 27 ° C)

The development is colorless to pale yellow [2 cb, Ivory Tint], poor formation of aerial hyphae, and partially develops a slight white aerial hyphae after 3 weeks of culture. No soluble dye can be determined.

(5) Yeast-malt agar medium (ISP—medium 2) (27 ° C culture)

White aerial mycelium develops slightly on the development of colorless to pale yellow [2 gc, Bambo]. No soluble dye is found.

(6) Automated agar medium (ISP-medium 3) (cultured at 27 ° C)

Colorless to light yellow [2ca, Lt1 vory], and on the development of light pink [5ec, DustyPeach] aerial hyphae, no soluble pigment is observed.

3. Physiological properties

(1) Suitable temperature range for growth

Using yeast starch agar medium (soluble starch 1.0%, yeast extract 0.2%, string agar 2.4%, H7.0) at 10 ° 20 ° C, 24. Tests at temperatures of C, 27 ° C, 30 ° C, 37 ° C, and 50 ° C have shown that they grow at any of these temperatures other than 10 ° C and 50 ° C. The optimum growth temperature is considered to be around 30 ° C.

(2) Starch hydrolysis (Starch 'inorganic salt agar medium, ISP—medium 4, 27 ° C culture)

About 5 days after cultivation, starch hydrolysis began to be observed, and its hydrolysis activity was moderate.

(3) Melanin-like pigment formation (Tryptone 'Yeast' broth (ISP-medium) 1), peptone 'yeast' iron agar medium (ISP medium 6), tyrosine agar medium (ISP-medium 7), all at 27 ° C

, Is negative even in the shifted medium.

(4) Utilization of carbon source (Pridham's Gotrib agar medium (ISP—medium 9), culture at 27 ° C)

It grows using D-glucose, L-arabinose, D-xylose, rhamnose and raffinose. Inositol will probably make use of it. Do not use D-fructoses, sucrose, or D-mannitol.

(5) Nitrate reduction reaction (0.1% nitric acid lipe containing eptone water (ISP medium-8) at 27 ° C)

Negative.

(6) Liquefaction of gelatin (15% simple gelatin medium, culture at 20 ° C; glucose, peptone, gelatin medium, culture at 27 ° C)

In the case of the 15% simple gelatin medium, the growth at 20 ° C was poor, and the liquefaction was not observed. In the case of glucose 'peptone' gelatin medium, the power at which gelatin liquefaction starts around day 18 after culturing The effect is extremely weak. However, in this case, a retest is required.

(7) Coagulation of skim milk, peptone conversion (Skim milk powder, culture at 37 ° C)

Peptone formation starts around 10 days after cultivation without coagulation, and its effect is weak to moderate.

Summarizing the above properties, the MK251-43F3 strain elongates a spiral-forming aerial mycelium from a branched basal hypha and produces 10 to 50 oval ovules in the mature spore chain. Spores, and its surface is spiny to hairy. In various media, the growth is colorless to light yellow and aerial mycelium may not form, but in some media white to brown to light pink. No soluble dye is produced. The optimum growth temperature is around 30 ° C. Melanin-like pigment formation is negative, starch hydrolyzes moderately, and proteolytic activity needs to be retested, but weak to moderate. The 2,6-diaminopimelic acid contained in the cell wall was of the shishi type. The bacterial component menaquinone mainly contains MK-9 (H6) and also contains MK-9 (H8). From these properties, the MK 25 1 -43 F 3 strain is Streptomyces

It is considered to belong to the genus (Strep t omy c e s). A search for closely related known strains revealed that Streptomyces flaveolus (S treptomyces cesfl ave olus) (literature literature). P. 1968) and Streptomyces fasicrateus

(S t r e p t omy c e s f l av e o l u s) (Reference

I nternati on al J our nalof Sy stemmatic Bacterio 1 ogy, Vol. 18, p. 108, p. 1968) _{c. The} above two conserved strains and MK 251-43 F3 strain At present, the MK251-143F3 strain is Streptomyces sp.

(Str e p t omy c e s s p.) MK 25 1—43 F3.

The MK 25 1-43 F3 strain was applied to the Institute of Biotechnology, Institute of Industrial Science and Technology (1-3, Higashi 1-3-chome, Tsukuba-shi, Ibaraki Prefecture), and the deposit number FE RM P. — Deposited as No. 1 6065 and entered into the International Deposit No. FERM BP—657 1 under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Patent Procedure on January 11, 1998 Transferred.

The strain belonging to the genus Streptomyces used in the present invention, like other strains of the genus Streptomyces, is liable to change its state of life, for example, by artificial mutation using ultraviolet rays, X-rays or chemicals. Although it can be easily mutated, any mutant can be used in the present invention as long as it has the ability to produce the physiologically active substance sulfostin of the present invention.

In order to produce sulfostine, the above strain is first aerobically cultured in a medium containing a nutrient that can be used by actinomycetes. As the nutrient source, known nutrients conventionally used for cultivation of actinomycetes can be used. For example, as a carbon source, glucose, fructose, glycerin, sucrose, dextrin, galactose, organic acids and the like can be used alone or in combination.

Sources of inorganic and organic nitrogen include ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, peptone, meat extract, yeast extract, dried yeast Mother, corn 'Steep' liquor, soy flour, cottonseed oil scum, casamino acid, baked soy ton, soluble vegetable. Protein, protein, etc. can be used alone or in combination.

In addition, if necessary, inorganic salts such as salt, calcium carbonate, magnesium sulfate, copper sulfate, iron sulfate, zinc sulfate, manganese chloride, and phosphate can be added to the medium. Further, organic substances such as amino acids, vitamins, nucleic acids and inorganic substances can be appropriately added to the medium.

The most suitable culture method is a liquid culture method, particularly a deep stirring culture method. The culture temperature is from 20 ° C to 45 ° C. It is desirable to culture in slightly acidic or slightly alkaline pH.

In liquid culture, sulfostine substances are usually produced and accumulated in the culture solution after culturing for 3 to 8 days. When the amount of sulfostine produced in the cultured cells reaches the maximum, stop the culture, filter the cells and the culture solution, and purify and isolate the desired product from the cells. For the purification and isolation of this substance from bacterial cells, a separation and purification method generally used to isolate a metabolic product of microorganisms from the cultured bacterial cells is used.

For example, the culture solution (15 L) is separated into the filtrate and the cells by the usual filtration method. The obtained filtrate is degassed and stirred with activated carbon equilibrated with water for 2 hours. From this mixture, an activated carbon non-adsorbed fraction is obtained by a usual filtration method. After concentrating the obtained fraction under reduced pressure, add 10 equivalents of ethanol, suspend, and filter to obtain an ethanol-insoluble fraction. Further, the ethanol-insoluble fraction is washed with methanol and dried under reduced pressure to obtain a methanol-insoluble fraction.

The methanol-insoluble fraction is subjected to microcrystalline cellulose column chromatography (Funacel, manufactured by Funakoshi Co.) developed with n-butanol-monoacetic acid-monohydrate (2: 1: 1) to obtain an active fraction. This active fraction was dried under reduced pressure and freeze-dried.

Chromatography on a microcrystalline cellulose column developed with n-butanoyl acetate monohydrate (3: 1: 1) yields the active fraction. The active fraction is dried under reduced pressure and freeze-dried to obtain a crude substance. This crude substance was dissolved in 0.2 N aqueous ammonium bicarbonate, and subjected to DEAE Sephadex A-25 column chromatography (Pharmacia) developed with 0.2 N aqueous ammonium bicarbonate in advance to separate the active fraction. obtain. this The active fraction is evaporated to dryness under reduced pressure, lyophilized, dissolved in 0.03 N aqueous ammonia bicarbonate, and developed using a Showdex IEC DE AE-825 column developed with 0.03 N aqueous ammonium bicarbonate in advance. Apply liquid chromatography (Pharmacia) to obtain sulfostine (0.5 mg).

The physicochemical properties of the physiologically active substance sulfostin obtained as described above are shown below.

1) Appearance: white powder

2) Molecular weight: 2 72

3) Molecular formula: C ₅ H, ₃ N ₄ Os SP

4) Solubility: soluble in water, insoluble in lower alcohols, acetone, ethyl acetate, hexane, petroleum ether,

5) R f value by silica gel thin layer chromatography: developing solvent of n-butanol monoacetic acid mono water (2: 1: 1) shows 0.28,

6) Ultraviolet absorption spectrum: showing terminal absorption,

7) Infrared external absorption spectrum: shows the spectrum measured with the bromide-resistant pill tablet shown in the attached Fig. 1, and specifically shows the following characteristic absorption band (cm ¹ ): 351 0, 3 3 5 5, 3 2 5 0, 1 6 72, 1 6 5 8, 1 6 24, 1 5 4 5, 1 3 6 5, 1 3 0 8, 1 2 0 0, 1 1 8 8, 1 1 3 0, 1 0 6 4, and 9 2 2,

8) Hydrogen nuclear magnetic resonance spectrum: shows the spectrum measured in heavy water shown in Fig. 2 attached, and specifically shows the following signal (5 (p pm): 4.18) (1H, dd, J2 6.8, 12.0 Hz), 3.82 (1H, tdd, J = 5.1, 7.3, 13.0Hz), 3.70 ( 1 H, tdd, J = 5.1, 6. 7, 1 3.0 Hz), 2.39 to 2.44 (1 H, m), 2.10 to 2.28 (1 H , M), and 1.89 to 2.03 (2 H, m),

9) Carbon nuclear magnetic resonance spectrum: shows the spectrum measured in heavy water shown in Fig. 3 attached, and specifically shows the following signal 5 (pm): 20.5, 24 2, 45.4, 51.3, and 12.4,

10) Phosphorus nuclear magnetic resonance spectrum: shows the spectrum measured in heavy water as shown in the attached Figure 4, specifically shows the following signal <5 (ppm): 6.01, 1 1) Specific rotation: [a] D ⁱⁱ -2 1.5 degrees (c 0.52, H ₂₀ ),

1 2) Melting point: 203-208 ° C (decomposition),

13) Color reaction: Positive for ninhydrin reaction and Lydon's-Smith reaction.

Various methods known in the art can be used for preparation and administration when used as pharmaceuticals. Administration can be by injection, oral, rectal, etc. The preparation may take the form of injections, powders, granules, tablets, suppositories and the like. Various adjuvants used for pharmaceuticals, i.e., carriers and other auxiliaries, such as stabilizers, preservatives, soothing agents, emulsifiers, etc., as required, as long as they do not adversely affect sulfostine during formulation Can be used. The sulfostine content of the preparation can be varied over a wide range depending on the form of the preparation and the like. Generally, the preparation contains 0.01 to 100% by weight, preferably 0.1 to 70% by weight of sulfostin. The balance includes carriers and other auxiliaries usually used for pharmaceuticals.

The dosage of sulfostine varies depending on the symptoms and the like, but is about 0.01 to 80 mg per adult per day. If continuous throwing is required, it is preferable to reduce daily usage.

The inhibitory effect of sulfostine on dipeptidyl peptidase IV will be described below with reference to experimental examples.

Experimental Example 1 Measurement of Dipidyl Peptidase IV Activity

The measurement was carried out as follows in accordance with the literature (Bi0 chem. Biopsys. Acta, 25, 58, 91 to 59, 9 (1972)). 3.2 mM glycyl'-puril / 3-naphthylamide (Bachem, Switzerland) 0.025 ml and 0.1 M Tris-maleic acid buffer (PH7.2) 0.1 Drug in 0.1 ml , And water was added so that the final volume became 0.2 ml, to prepare a mixed solution. 37 for the mixed solution. C, warm for 10 minutes, add 0.025 ml of dipeptidyl peptidase IV solution partially purified from rat kidney homogenate by ammonium sulfate fractionation to the above mixed solution, and add Allowed to react for hours. After the reaction, the reaction mixture contains 10% polyoxyethylene sorbitan monolaurate (addition of 20 mol of ethylene oxide) and 0.2% fast garnet GBC salt (Sigma, USA). The reaction was stopped by adding 0.1 ml of sodium buffer (DH 3.78), and the At (a) was measured. At the same time, the absorbance (b) of a blind test using only a buffer solution containing no sample was measured, and the diptidylpeptidase IVP and the harm rate were calculated using the following formula.

[(b a) / b] x 1 0 0

Was calculated by Table 1 shows the dipeptidyl peptidase IVP and harmful activity values of the compounds of the present invention measured by this method.

table 1

Thus, sulfostine has a strong inhibitory effect on dipeptidyl peptidase IV, and its IC ₅ . The value was 0.0082〃g / m1.

Industrial applicability

The novel physiologically active substance sulfostin of the present invention can be used for medicaments such as immunomodulators, hormonal regulators, anti-HIV drugs, anti-allergic drugs, anti-inflammatory drugs, and anti-rheumatic drugs.

Claims

The scope of the claims -

1. Sulfostine, a physiologically active substance with the following physicochemical properties:

1) Appearance: white powder,

2) Molecular weight: 272,

3) Molecular formula: C ₅ H, ₃ N ₄ Os SP,

5) R f value by silica gel thin-layer chromatography: n-butano-l-acetic acid mono-water (2: 1: 1)

6) Ultraviolet absorption spectrum: showing terminal absorption,

7) Infrared absorption spectrum: shows the following characteristic absorption bands (cm— ') when measured on a potassium bromide tablet: 3510, 3355, 3250, 16 72, 1658, 1624, 1654, 1365, 1308, 1240, 1188, 1130, 1064, and 9222 ,

8) Hydrogen nuclear magnetic resonance spectrum: shows the following signal <5 (ppm) when measured in heavy water: 4.18 (1H, dd, J = 6.8, 12.0Hz) , 3.82 (1H, tdd, J = 5.1, 7.3, 13.0 Hz), 3.70 (1H, tdd, J = 5.1, 6.7, 1 3.0 Hz), 2.39 to 2.4.4 (1H, m), 2.10 to 2.28 (1H, m), and 1.89 to 2.03 (2 H, m),

9) Carbon nuclear magnetic resonance spectrum: shows the following signals <5 (ppm) when measured in heavy water: 20.5, 24.2, 45.4, 51.3, and 1 72.4,

10) Phosphorus nuclear magnetic resonance spectrum: shows the following signal δ (pm) when measured in heavy water: 6.0 K

1 1) Specific rotation: [α],) ² "— 21.5 degrees (c 0.52 Η ₂₀ ),

1 2) Melting point: 203 ~ 208 ° C (decomposition),

13) Color reaction: Positive for ninhydrin reaction and Lydon-Smith reaction.

2. Sulfostine, a physiologically active substance with the following physicochemical properties:

1) Appearance: white powder, 2) Molecular weight: 2 7 2,-

3) Molecular formula: Cr, _{Hi 3} N ₄ Or, SP,

5) Rf value by silica gel thin layer chromatography: developing solvent of n-butanolmonoacetic acid-water (2: 1 _: 1) shows 0.28,

6) Ultraviolet absorption spectrum: showing terminal absorption,

7) Infrared absorption spectrum: shows the spectrum measured with the potassium bromide tablet shown in Fig. 1 attached.

8) Hydrogen nuclear magnetic resonance spectrum: shows the spectrum measured in heavy water shown in Fig. 2 attached.

9) Carbon nuclear magnetic resonance spectrum: shows the spectrum measured in heavy water shown in Fig. 3 attached.

10) Phosphorus nuclear magnetic resonance spectrum: shows the spectrum measured in heavy water shown in the attached Figure 4.

1 1) Specific rotation: []. ²⁸ - 2 1.5 ° _{(c 0. 5 2, H 2} 0),

1 2) Melting point: 203 ~ 208 ° C (decomposition),

13) Color reaction: Positive for ninhydrin reaction and Lydon's-Smith reaction.

3. A microorganism belonging to the genus Streptomyces and having the ability to produce the physiologically active substance sulfostine according to claim 1 or 2 is cultured to produce and accumulate the physiologically active substance sulfostin in the culture. A method for producing a physiologically active substance sulfostin, which comprises collecting spores from a culture.

4. A dipeptidyl peptidase IVP containing the physiologically active substance sulfostine according to claim 1 or 2 as an active ingredient.

5. A medicament comprising the physiologically active substance sulfostine according to claim 1 or 2 as an active ingredient c 6. Streptomyces ′ SP MK2 5 1—4 3 F 3 (S trept omy cess) having the ability to produce the physiologically active substance sulfostin p. MK 2 5 1-4 3 F 3) or a mutant thereof.